Colorectal cancer is the third leading cause of cancer with more than half of cases occurring in advanced stages that are difficult to cure. New prevention strategies are needed. The colonic microbiome plays an important role in the etiology of colorectal cancer (CRC). Our laboratory has studied Enterococcus faecalis in models of inflammation-associated CRC. This human commensal can polarize (or activate) macrophages to reprogram and transform epithelial cells into cancer cells. This process is referred to as the ?microbiome-induced bystander effect.? In CRC, tumor stem-like cells are believed to originate from stem cells at the base of crypts. However, newer data supports fully differentiated epithelial cells at the top of crypts as cells of origin for CRC. This fits with a ?top-down? model of morphogenesis for human colon adenomas in which transformed stem-like cells expand downward to replace normal crypt structures. In this project, we hypothesize that microbiome-polarized colon macrophages cause mutagenesis and reprogramming of differentiated epithelial cells in the upper portion of crypts toward pluripotency and stemness, thereby driving malignant transformation. Using established in vitro and in vivo models for microbiome-triggered and macrophage-induced malignant transformation, we will assess differentiated colon epithelial cells for reprogramming and mutation during transfor- mation into tumor stem-like cells. Mutations in key driver genes along with genome-wide changes in chromosomes, gene expression, and methylation will be assessed to establish the sequence of events during reprogramming and transformation. Finally, transformed malignant clones will be enriched by organoid culture and allografts characterized in immunodeficient mice. The preventive effect of probiotics (Lactobacillus and Bifidobacterium) on a microbiome- induced bystander effect will also be assessed. Using these same in vitro and in vivo models, the role of signaling by tumor necrosis factor alpha as a key mediator for the microbiome- induced bystander effect will be assessed. We hypothesize that blocking a receptor for tumor necrosis factor alpha will lead to downregulation of a functional tumor stem-like cell marker, doublecortin-like kinase 1, and thereby prevent microbiome-driven colon carcinogenesis. These experiments will help determine how intestinal commensals polarize innate immune cells to produce bystander effects and thereby induce mutagenesis, reprogramming and transformation of colon epithelial cells. Finally, we will investigate whether probiotics can inhibit microbiome- driven carcinogenesis and thereby bolster this as an approach to CRC prevention.